1. Field of the Invention
This invention pertains to improved new membranes for use in connection with biological assays performed on membranes, specifically, chemiluminescent assays where the analyte is deposited on a membrane, and subjected to subsequent processing, the processing involving indicating the presence or absence and quantity of a suspected element in the analyte by the release of chemiluminescence. More specifically, this invention pertains to improved membranes which can be used in connection with chemiluminescent assays based on 1,2-dioxetanes which can be triggered by enzymes and enzyme conjugates in ligand-binding pairs to emit light.
2. Background of the Prior Art
Increasingly, blotting assays employing chemiluminescent detection have become a popular modality for the detection of proteins and nucleic acids. Conventionally, such assays are conducted by isolating a sample of the analyte on a membrane, exposing that membrane to an antibody-agent or nucleic acid probe agent complex, wherein the agent causes a composition to be added to undergo chemiluminescence. In its most widely practiced form, this type of blotting assay employs an enzyme or enzyme conjugate as the agent causing a compound which can decompose to release light to undergo that decomposition, thereby giving chemiluminescence. Among the most popular compounds for this purpose are 1,2-dioxetanes. These structures, if stabilized with an adamantyl group or similar stabilizing group, or derivatized adamantyl group, can be protected with an enzyme-labile group which, when cleaved by a suitable enzyme attached to the antibody or nucleic acid probe secured to the target compound of the analyte, forms an unstable anion, which then decomposes to release light. In this case, using an enzyme, the chemiluminescent compound is a substrate, and among dioxetane substrates, AMPPD disodium 3-(4-methoxy-spiro[1,2-dioxetane-3,2'-tricyclo[3.3.1.1.sup.3,7 ]decan]-4-yl) phenyl phosphate is widely used. Structurally related compounds, wherein the adamantyl group is substituted with various electron-active groups, convert the adamantyl moiety from a mere stabilizing agent to one which actively influences dioxetane decomposition. Among these, the chlorine-substituted compound, or CSPD, has been demonstrated to be markedly effective. A wide variety of other compounds, bearing other enzyme-labile protective groups, such as sugar, acetate and other ether and ester moieties are known and effective.
The protocol used in such blotting assays is conventional, and among various blotting assays, Western Southern Blotting are widely known. In such assays, proteins (or in related assays, nucleic acids) are purified, and transferred to membrane supports. Generally known membranes include nitrocellulose, nylon, PVDF and others. This transferred material (analyte) is incubated with at least one antibody specific for the compound being sought (specific protein or nucleic acid). In a Western Blotting assay the antibody can be complexed with an enzyme, or, a second antibody, complexed with an enzyme, can be added following a washing step. In the case of AMPPD and CSPD, the binder (antibody or DNA/RNA probe) is conjugated with an alkaline phosphatase enzyme. Subsequent to washing, the blot is incubated with the chemiluminescent substrate. Release of chemiluminescence is confirmation of the presence of the suspected compound or target analyte.
In Southern blotting procedures nucleic acid sample is blotted onto a membrane following gel electrophoresis separation. Hybridizations are performed with enzyme labeled nucleic acid probes (labiled directly or indirectly via biotin-avidin or antibody-antigen bridge) containing base sequence complementary to regions specific for the target sample. Again, subsequent to washing, the blot is incubated with the chemiluminescent substrate and the subsequent release of light signal is confirmation of the presence of the suspected nucleic acid sequence.
This blotting format presents certain problems in connection with the membrane supports identified. The chemical content of the membrane surface, to which the chemiluminescent substrate is exposed, has a tendency to quench or promote quenching of the emitted light, thus reducing the intensity of the chemiluminescent signal. Further, the membranes used have significant lot-to-lot variations, due to current production processes. As a result, it is difficult both to standardize the process, and to provide for automatic data acquisition. Among specific problems encountered are low signal levels, very high nonspecific backgrounds, and membrane-initiated decomposition of chemiluminescent substrates, such as AMPPD and CSPD.
When dealing with dioxetane substrates such as AMPPD, it is important to note that these compounds have very low intensities of chemiluminescence in aqueous, protic environments. This is believed to be due principally to proton transfer quenching reactions, or dipole-dipole interactions which tend to promote dark reactions of the excited state ultimately produced by enzyme cleavage. Proton transfer reactions are extremely well known in organic chemistry, and can easily compete with light emission during the lifetime of the excited state, which is several orders of magnitude slower. Shizuka, "Accounts of Chemical Research", 1985, Vol. 18, pages 141-147. This can be confirmed by the case that the chemiluminescent efficiency of AMPPD in aqueous buffers is approximately only 10.sup.-6, but improves, in the presence of a hydrophobic medium, by approximately 10.sup.4.
In addition to the above-noted problems, conventional blotting assays continue to leave certain goals unmet. Of particular importance is the ability to quantitate the levels of nucleic acid fragments, or proteins, identified in blotting applications. Currently, blotting assays are qualitative in nature, confirming only the presence or absence of the component sought for. Frequently, a component will be present in all analytes, but diagnosis of a disease depends on the level of the component in the analyte. Current blotting techniques do not permit discrimination on this basis.
Another unmet goal of blotting assays is the provision of membranes which will permit sharply resolved bands, corresponding to bound component, in the absence of background, which would be suitable for automated data acquisition. As an example, scanning charged-coupled devices can be employed in reading complex information such as DNA sequences. Such automation would permit higher efficiency of error-free data acquisition. Current blotting assays based on chemiluminescent compounds such as dioxetanes do not provide the necessary sharp resolution of bands or high intensity signal in the absence of background to permit automated data acquisition.
Accordingly, it remains a goal of those of skill in the art to obtain membranes which can be used for chemiluminescent blotting assays, based on enzyme-triggerable dioxetanes, to provide improved, quantifiable information.